GSM8047388: RNAseq_U2OS_G3BPKO_DMSO_Rep1; Homo sapiens; RNA-Seq
Cells at 80-90% confluency were harvested from a 15 cm dish. Cells were incubated with 1 µM thapsigargin for 2hrs, and then treated with 50 µg/mL cyclohexamide (CHX) for 1 minute. Then, cells were washed gently with PBS containing 50 µg/mL CHX and lysed in ice-cold lysis buffer (20mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% v/v Triton x-100, 1mM DTT, 20-25 U/mL TURBO DNAse (Thermo Fisher Scientific, AM2238), 100 µg/mL CHX). Cells were triturated with a 26-gauge needle. Lysate was recovered after spinning for 10 min, 20,000g, at 4°C. Total RNA was extracted from 25 µL of lysate with a Direct-zol kit. rRNA was depleted with a NEBNext rRNA Depletion Kit (NEB, E7400), and following manufacturer's instructions. Libraries were prepared with a NEBNext Ultra II Directional RNA Library Prep Kit of Illumina (E7760), and following manufacturer's instructions. cDNA libraries were amplified with multiplex oligos for Illumina sequencing (NEB, E6609S).